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In vivo Glut4 translocation in lean and fatty Zucker rat hearts: effect of insulin dose and time

University of British Columbia

Glucose transporter type 4 (Glut4) is an isoform of facilitative glucose transporters which plays an important role in whole body glucose homeostasis. Glut4 translocation to the plasma membrane from intracellular compartments is regulated by insulin in insulin-sensitive tissues including the heart. Cardiac insulin resistance in the obese (fa/fa) Zucker rat has been characterized by depressed glucose utilization as well as glucose transport. Glucose transporter translocation has been assessed in the insulin resistant heart by measuring total glucose transporters and was shown to be responsive to insulin under in vitro conditions. The aim of the present study was to examine specifically the translocation of the Glut4 isoform in the heart of obese rats under in vivo conditions. The insulin dose effect and the time course of cardiac Glut4 translocation in vivo was examined in lean (FA/?) and fatty (fa/fa) Zucker rats by determining the subcellular distribution of Glut4. Plasma membrane and intracellular membrane fractions were purified from the heart ventricles isolated from rats either in the basal state or injected with insulin. Glut4 content in the membrane fractions was determined using a competitive ELISA method. Insulin injection, given at doses 2.5, 5 and 10 Ukg, increased plasma insulin levels in both lean and fatty Zucker rats when compared to the basal level of each phenotype (p<0.05, one-way ANOVA). Insulin resistance in the fatty Zucker rat was shown by the inability of all three doses of insulin to significantly reduce plasma glucose (p>0.05, one-way ANOVA) in spite of the elevation in plasma insulin levels. On the other hand, plasma glucose was significantly reduced with all three insulin doses in lean Zucker rats (p<0.05, one-way ANOVA). At the highest insulin dose and at 20 minutes post insulin injection, Glut4 recruitment to the plasma membrane in lean rats was shown by a significant increase (30%) in plasma membrane Glut4 content when compared to its basal level (p<0.05, two-way ANOVA). In fatty rats, Glut4 mobilization to the plasma membrane was shown to be similarly responsive to insulin as shown by a significant and similar increase (39%) in the Glut4 content (p<0.05, twoway ANOVA). However, plasma membrane Glut4 content in the fatty Zucker rat was shown to be significantly lower than the lean phenotype in both the basal state and insulin-stimulated state (p<0.05, two-way ANOVA). In the same insulin-stimulated state, Glut4 mobilization from the intracellular membrane was shown to be responsive to insulin in the lean Zucker rat but not in the fatty Zucker rat. This was shown by a significant reduction in the Glut4 content (37%) in the lean group (p<0.05, two-way ANOVA) and no significant drop in fatty Zucker rats (p>0.05, two-way ANOVA). In the basal state, intracellular Glut4 content in fatty Zucker rats was significantly lower than lean controls (p<0.05, two-way ANOVA). The effect of lower insulin doses on cardiac Glut4 translocation was examined and dose response curves for Glut4 mobilization to the plasma membrane and from the intracellular membrane were constructed for both lean and fatty Zucker rats. It was shown that insulin doses, at 2.5, 5, and 10 U/kg, significantly recruited Glut4 to the plasma membrane with the maximal response obtained at a dose of 2.5 U/kg for each phenotype (p<0.05, one-way ANOVA). In lean Zucker rats, Glut4 recruitment to the plasma membrane with all three insulin doses was associated with Glut4 mobilization from the intracellular store as shown by significant reductions in the Glut4 content from the basal level (p<0.05, one-way ANOVA). However in the fatty Zucker rat, Glut4 content in the intracellular membrane fraction was not significantly reduced with all three insulin doses (p>0.05, one-way ANOVA). The time profile of cardiac Glut4 translocation showed that maximal response was already reached at 5 minutes after insulin injection and persisted for 25 minutes, as shown by no significant differences in Glut4 content among the various time points within the 25-minute period in each of the membrane fractions (p>0.05, one-way ANOVA). The time profile of transporter translocation further substantiated the observation that insulin was able to induce Glut4 mobilization to the plasma membrane in the fatty Zucker rat without changing Glut4 content in the intracellular membrane fraction. Evaluation of the membrane fractions using Na⁺/K⁺ ATPase, a marker enzyme for the plasma membrane, showed high enrichment (88 fold) of the enzyme in the plasma membrane fraction when compared to the homogenate. Less than 15% contamination of plasma membrane was found in the intracellular fraction. In the fatty Zucker rat, there was a 40% reduction in the Na⁺/K⁺ ATPase activity in the plasma membrane fraction when compared to the lean phenotype. These results may suggest an overall decrease in the enzyme activity in the insulin resistant heart, or may suggest a difference in the purity of plasma membrane fractions obtained between the lean and fatty rat. Taken all together, the results obtained from the current study showed a lower Glut4 content in both the plasma membrane and intracellular membrane in the fatty Zucker rat heart which may suggest an overall decrease in the cardiac expression of Glut4. Cardiac Glut4 translocation in the fatty rat, as shown by recruitment of the protein to the plasma membrane, was found to be similarly responsive as the lean control. However, Glut4 mobilization to the plasma membrane in the fatty Zucker rat was shown to be not associated with a reduction in Glut4 content in the intracellular membrane fraction. This observation is not understood at present and further experiments are required to confirm this finding.

Thesis/Dissertation

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2009-02-12

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http://hdl.handle.net/2429/4540

Pharmacology

University of British Columbia

UBCV

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